CN102343133A - Microbial degradation method of nicotine insecticide thiacloprid - Google Patents
Microbial degradation method of nicotine insecticide thiacloprid Download PDFInfo
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- CN102343133A CN102343133A CN2011101456836A CN201110145683A CN102343133A CN 102343133 A CN102343133 A CN 102343133A CN 2011101456836 A CN2011101456836 A CN 2011101456836A CN 201110145683 A CN201110145683 A CN 201110145683A CN 102343133 A CN102343133 A CN 102343133A
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- thiophene worm
- worm quinoline
- thiacloprid
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Abstract
The invention relates to microbial degradation technology of nicotine insecticide thiacloprid and an application in microbial synthesis of thiacloprid amide. The method comprises the following steps: inoculating microbe which can degrade thiacloprid in a culture medium, culturing at 20-40 DEG C for 10-48 hours, washing, collecting bacteria; adding the bacteria into a buffer which contains the degradation substrate of 10-200 mg/l of thiacloprid and has a pH of 6.0-8.5, performing shaking culture at 20-40 DEG C; performing thiacloprid degradation and converting thiacloprid into thiacloprid amide. The technology is simple in operation, and is applicable to the treatment of thiacloprid-containing waste water and sewage, or the remediation of thiacloprid-contaminated soil, and the biosynthesis of thiacloprid amide products.
Description
Technical field
The present invention relates to a kind of can be used for nicotinic insecticide thiophene worm quinoline microbial degradation technology and the application in the biosynthesis of acid amides thiophene worm quinoline thereof.
Background technology
The chloro nicotinic insecticide is to be used to prevent and treat the most effectively one of pesticide active ingredient of piercing mouth parts and some biting mouth parts insects.(Thiacloprid is a kind of sucking insect to be had broad spectrum pesticide efficiently Fig. 1) to thiophene worm quinoline, and the important pests on pomaceous fruits fruit, cotton, vegetables and the potato is had excellent biologically active.Except that to aphid and aleyrodid effectively, thiophene worm quinoline is also effective to various beetles and lepidoptera pest.At present, thiophene worm quinoline uses on global 50 multiple kinds of crops.Began to promote and use in China in recent years.The environmental behaviour research of thiophene worm quinoline shows that thiophene worm quinoline can be by hydroxylating in animal and plant body, dehydrogenation, hydrolysis etc.The main metabolic pathway of thiophene worm quinoline in soil is that hydrolysis cyanoimino pharmacophoric group generates acid amides thiophene worm quinoline.The applicant belongs to the laboratory and has reported a strain rhodotorula
Rhodotorula mucilaginosaIM-2 degradable thiophene worm quinoline also generates product acid amides thiophene worm quinoline, yet
R.
MucilaginosaThe auxocyte of the IM-2 thiophene worm quinoline (Dai et al. 2010) of need in adding the mineral salts medium of glucose, could degrading.People such as Ge Feng have applied for that (patent publication No.: CN101792439A), yet this method need be used strong acid and highly basic and reaction medium acetone for a kind of patent of invention of chemical synthesis process of acid amides thiophene worm quinoline.
The present invention adopts the non-auxocyte of microorganism (resting cell) to carry out the degraded and the synthesizing amide thiophene worm quinoline of thiophene worm quinoline, does not need HTHP, method gentleness and safer.In thiophene worm quinoline degraded and synthesizing amide thiophene worm quinoline process, need not add the common metabolic degradation that primary energy material such as glucose is used for thiophene worm quinoline.Thereby this technology has, and technology is simple, characteristics such as economize in raw materials.
Summary of the invention
The purpose of this invention is to provide a kind of method that primary energy material such as glucose is used for the common metabolic degradation of thiophene worm quinoline of in thiophene worm quinoline degraded and synthesizing amide thiophene worm quinoline process, need not adding.
Principle of the present invention is to adopt microbial degradation to carry out the degraded and the synthesizing amide thiophene worm quinoline of thiophene worm quinoline as shown in Figure 1.
The present invention cultivates earlier and obtains microbial cells, and thalline is joined in the cushioning liquid that contains finite concentration thiophene worm quinoline, takes a sample behind the certain hour, and high performance liquid chromatography (HPLC) is analyzed the concentration of thiophene worm quinoline.Compare with the control group that does not add thalline like thiophene worm quinoline concentration, obvious reduction is arranged, represent that then this microbial strains has the degradation capability of thiophene worm quinoline.Adopt said method, the present invention screens from strain library
Variovorax paradoxusCGMCC 1.1842;
Acinetobacter johnsoniiACCC 02175;
Ochrobacterium anthropiCGMCC 1.2501;
Sinorhizobium medicaeCGMCC 1.2547 bacterial classifications such as grade have thiophene worm quinoline degradation capability.The cyanic acid of thiophene worm quinoline changes amide groups in the degradation process, generates acid amides thiophene worm quinoline.
The microbial degradation method of nicotinic insecticide thiophene worm quinoline of the present invention, thalline is washed and collected to the microorganism that is inoculation degradable thiophene worm quinoline behind 20-40 ℃ of cultivation 10-48h, in nutrient solution; Thalline is joined in the buffer solution of the pH6.0-8.5 that contains degraded substrate 10-200mg/L thiophene worm quinoline, in 20-40 ℃ of shaken cultivation.Utilize the metabolic capability of microorganism to carry out the degraded of thiophene worm quinoline and the biosynthesis of acid amides thiophene worm quinoline simultaneously.This method technology is simple and safe, easy and simple to handle, can be used for the sewage disposal of thiophene worm quinoline and the biosynthesis of contaminated soil reparation of thiophene worm quinoline and acid amides thiophene worm quinoline.
Above-mentioned bacterial strain with thiophene worm quinoline degradation capability can be
Variovorax paradoxusCGMCC 1.1842;
Acinetobacter johnsoniiACCC 02175;
Ochrobacterium anthropiCGMCC 1.2501;
Sinorhizobium medicaeCGMCC 1.2547 bacterial classifications such as grade.Above-mentioned bacterial classification can buy in culture presevation administrative center.
The above-mentioned nutrient solution that is applied to the thalline cultivation has no particular limits, and can be any nutriment that is suitable for the mentioned microorganism growth.
The mentioned microorganism fermentation culture conditions is: 20-40 ℃, 50-300 rpm shakes bottle vibration ventilation or stirs ventilation oxygen-supplying, fermentation 10-48h.
Mentioned microorganism need not add special derivant induced degradation enzyme and acid amides thiophene worm quinoline synzyme during the fermentation.
Above-mentioned zymotic fluid separates with thalline, can adopt centrifugal separation method or filter separation method to obtain somatic cells.
The condition of above-mentioned degraded thiophene worm quinoline does; The concentration of substrate thiophene worm quinoline is between 10-200mg/L; Degrade in the cushioning liquid and synthesizing amide thiophene worm quinoline at any one pH 6.0-8.5; Temperature range is at 20-40 ℃; 50-300 rpm shakes bottle vibration ventilation or stirs ventilation oxygen-supplying, and the reaction time is between 10-60h.
Description of drawings
Fig. 1 microbial degradation thiophene worm quinoline generates product acid amides thiophene worm quinoline.
Fig. 2
Variovorax paradoxusCGMCC 1.1842 degraded thiophene worm quinolines and generation acid amides thiophene worm quinoline curve.
Fig. 3
Variovorax paradoxusThe HPLC and the mass spectral analysis of CGMCC 1.1842 degraded thiophene worm quinolines.
The HPLC that Fig. 4 does not add the thiophene worm quinoline contrast of thalline analyzes.
It is right that Fig. 5 adds glucose
Variovorax paradoxusThe HPLC of CGMCC 1.1842 degraded thiophene worm quinoline influences analyzes.
The specific embodiment
Embodiment 1. will
Variovorax paradoxusCGMCC 1.1842 lines on the LB solid medium, cultivates 36h in 30 ℃.With 200 mL LB nutrient solutions, the 500 mL triangular flasks of packing into; Behind 121 ℃ of high pressure steam sterilization 15 min; After being cooled to room temperature; Be set forth in the bacterial classification of growing on the solid medium in the access; 30 ℃; Behind the 200 rpm oscillation and fermentation cultivation 24h; Stop fermentation, 5000rpm is centrifugal, and the thalline that makes separates with zymotic fluid, after the phosphate buffer washing of thalline with pH7.0; Being suspended in cumulative volume is in the 500mL triangular flask that contains 200 mg/L thiophene worm quinoline phosphate buffers (pH 7.0) of 200mL; 30 ℃, 200 rpm vibration ventilation is carried out the resting cell degraded and is transformed thiophene worm quinoline, behind the 60h; Stop reaction, centrifugal removal thalline.Thiophene worm quinoline content is reduced to 76.0mg/L in the HPLC detection supernatant; Acid amides thiophene worm quinoline content increases to 130mg/L (Fig. 2 and Fig. 3), and the thiophene worm quinoline concentration that does not add the control group of microbial cells does not then reduce (Fig. 3), does not detect the generation (Fig. 4) of acid amides thiophene worm quinoline simultaneously yet.The supernatant of centrifugal removal thalline is with equal volume of ethyl acetate twice, and the crystal that obtains after concentrating is removed substrate thiophene worm quinoline with the acetonitrile washing, can obtain 20mg purity and be 97% acid amides thiophene worm quinoline crystal.The carbon-13 nmr spectra data of acid amides thiophene worm quinoline are: 168.5,163.8,150.1,149.7,140.1,132.3,124.7,49.1,46.8,22.0; Hydrogen spectrum data are: 8.41 (d) 1H, 7.80 (dd) 1H, 7.47 (d) 1H, 6.54 (s) 1H, 6.26 (s) 1H, 4.70 (s) 2H, 3.51 (t) 2H, 3.01 (t) 2H.
Embodiment 2: identical with instance one, difference is to be contained in the buffer solution of thiophene worm quinoline and adds 2g/L glucose, and HPLC detects that thiophene worm quinoline content is reduced to 75.6/L in (Fig. 5) supernatant; Acid amides thiophene worm quinoline content increases to 128.4mg/L.Compare with the result of instance 1,
V. paradoxusCGMCC 1.1842 degraded thiophene worm quinolines and synthesizing amide thiophene worm quinoline do not need glucose as energy substance.
Embodiment 3: 200mL LB nutrient solution is put into the 500mL triangular flask, and 121 ℃ of high pressure steam sterilization 15 min insert
Acinetobacter johnsoniiACCC 02175 bacterial classification, 30 ℃, 50 rpm oscillation and fermentation cultivation; Behind the 10h; Stop fermentation, the centrifugal thalline that makes separates with zymotic fluid, and thalline adds in the 500mL triangular flask; And adding contains phosphate buffer (pH 8.5) 200mL of 150mg/L thiophene worm quinoline; 30 ℃, after 220rpm vibration ventilation transforms 48h, stop to transform; The centrifugal removal thalline of 8000 rpm, thiophene worm quinoline content reduces to 106mg/L by 150mg/L in the supernatant; Acid amides thiophene worm quinoline content increases to 36mg/L.
Embodiment 4: 300mL beef extract-peptone nutrient solution (pH7.0) is put into the 1000mL triangular flask, and 115 ℃ of high pressure steam sterilization 20 min after the cooling, insert
Ochrobacterium anthropi1.2501,30 ℃ of CGMCC, behind the 300rpm oscillation and fermentation cultivation 48h, centrifugal collection thalline.Dress 300mL contained in the 1000mL triangular flask of pH 6.0 buffer solutions of 200mg/L thiophene worm quinoline in thalline was suspended in, and carried out the degraded of thiophene worm quinoline in 30 ℃.Behind the 60h, sampling HPLC analyzes, and thiophene worm quinoline content is reduced to 170mg/L in the supernatant, and acid amides thiophene worm quinoline content increases to 22mg/L.
Embodiment 5: identical with instance 1, the bacterial classification of access does
Sinorhizobium medicaeCGMCC 1.2547.Sample analysis behind the 48h, thiophene worm quinoline content is reduced to 82mg/L by 200mg/L, and acid amides thiophene worm quinoline content increases to 103mg/L.
Embodiment 6: identical with instance 1, sample time is different.Sample analysis behind the 10h, thiophene worm quinoline content is reduced to 148mg/L by 200mg/L, and acid amides thiophene worm quinoline content increases to 50mg/L.
Embodiment 7: identical with instance 1, the thalline cultivation temperature is made as 20 ℃, and other conditions are identical, sample analysis, and thiophene worm quinoline content is reduced to 87.6mg/L by 200mg/L, and acid amides thiophene worm quinoline content increases to 112.5mg/L.
Embodiment 8: identical with instance 1.The thalline cultivation temperature is made as 40 ℃, and other conditions are identical.Thiophene worm quinoline content is reduced to 176mg/L by 200mg/L in the conversion fluid, and acid amides thiophene worm quinoline content increases to 18mg/L.
Embodiment 9: identical with instance 1.Thalline shakes a bottle rotating speed when cultivating and is made as 50rpm, and other conditions are identical.Thiophene worm quinoline content is reduced to 145.9mg/L by 200mg/L in the sample, and acid amides thiophene worm quinoline content increases to 51.6mg/L.
Embodiment 10: identical with instance 1.Thalline shakes a bottle rotating speed when cultivating and is made as 300rpm, and other conditions are identical.Thiophene worm quinoline content is reduced to 58.4mg/L by 200mg/L in the sample, and acid amides thiophene worm quinoline content increases to 143.6mg/L.
Embodiment 11: identical with instance 1.Shake a bottle rotating speed during degraded thiophene worm quinoline and be made as 50rpm, other conditions are identical.Thiophene worm quinoline content is reduced to 68.7mg/L by 200mg/L in the sample, and acid amides thiophene worm quinoline content increases to 133.2mg/L.
Embodiment 12: identical with instance 1.Shake a bottle rotating speed during degraded thiophene worm quinoline and be made as 300rpm, other conditions are identical.Thiophene worm quinoline content is reduced to 60.7mg/L by 200mg/L in the sample, and acid amides thiophene worm quinoline content increases to 139.6mg/L.
Embodiment 13: identical with instance 1.Substrate thiophene worm quinoline concentration is made as 10mg/L, and other conditions are identical.Thiophene worm quinoline content is by being reduced to 6 mg/L in the sample, and acid amides thiophene worm quinoline content increases to 3mg/L.
Embodiment 14: identical with instance 1.The temperature of degraded thiophene worm quinoline is 20 ℃, and other conditions are identical.Thiophene worm quinoline content is reduced to 80.1mg/L by 200mg/L in the sample, and acid amides thiophene worm quinoline content increases to 117.8mg/L.
Embodiment 15: identical with instance 1.The temperature of degraded thiophene worm quinoline is for being 40 ℃, and other conditions are identical.Thiophene worm quinoline content is reduced to 181.5mg/L by 200mg/L in the sample, and acid amides thiophene worm quinoline content increases to 17.7mg/L.
Claims (2)
1. the microbial degradation method of a nicotinic insecticide thiophene worm quinoline, it is characterized in that inoculating have degraded thiophene worm quinoline ability microorganism in nutrient solution, in 20-40 ℃ cultivate 10-48h after, washing and collect thalline; Thalline is joined in the buffer solution of the pH6.0-8.5 that contains degraded substrate 10-200mg/L thiophene worm quinoline, in 20-40 ℃ of shaken cultivation; Carry out the degraded of thiophene worm quinoline and simultaneously thiophene worm quinoline is converted into acid amides thiophene worm quinoline.
2. require the microbial degradation method of 1 described nicotinic insecticide thiophene worm quinoline according to power, it is characterized in that the microorganism with degraded thiophene worm quinoline ability is
Variovorax paradoxusCGMCC 1.1842;
Acinetobacter johnsoniiACCC 02175;
Ochrobacterium anthropiCGMCC 1.2501 or
Sinorhizobium medicaeCGMCC 1.2547.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108853865A (en) * | 2018-08-30 | 2018-11-23 | 广州大学 | A method of degradation dinotefuran |
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| CN1658759A (en) * | 2002-06-11 | 2005-08-24 | 纳幕尔杜邦公司 | Insecticidal amides with nitrogen-containing benzo-fused bicyclic ring systems |
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| CN1658759A (en) * | 2002-06-11 | 2005-08-24 | 纳幕尔杜邦公司 | Insecticidal amides with nitrogen-containing benzo-fused bicyclic ring systems |
| CN1993044A (en) * | 2004-08-06 | 2007-07-04 | 日本曹达株式会社 | Agricultural-chemical preparation having controlled releasability |
Non-Patent Citations (4)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108853865A (en) * | 2018-08-30 | 2018-11-23 | 广州大学 | A method of degradation dinotefuran |
| CN108853865B (en) * | 2018-08-30 | 2020-06-12 | 广州大学 | Method for degrading dinotefuran |
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Application publication date: 20120208 |